Statement of rights to inventions made under Federally-sponsored research: None
1. Field of the Invention
This invention pertains to the field of nucleic acid chemistry, and to combinatorial chemistry as it is applied to nucleic acids. More specifically, the invention concerns procedures for obtaining oligonucleotides and their analogs that can serve a function, most preferably as ligands, receptors, and catalysts. More specifically, this invention relates to methods for selecting in vitro oligonucleotide derivatives that can serve as ligands, receptors, or catalysts, starting from a pool of oligodeoxyribonucleotide derivatives that incorporate non-standard nucleobases, or incorporate standard nucleobases modified to carry additional functional groups, or standard oligonucleotides acting in conjunction with organic cofactors.
2. Background of the Invention
The majority of pharmaceutical agents are compounds that exert their biological activity by binding to a biological macromolecule, referred to here as a receptor. The discovery of ligands that can bind to a preselected receptor and thereby exert a biological effect is an important goal of medicinal chemists seeking to develop new human pharmaceutical agents.
Classically, ligands are discovered by three strategies. The first involves screening of a collection of chemicals whose structures have no deliberate connection with the structure or biology of the target receptor. This process is referred to as xe2x80x9crandom screeningxe2x80x9d.
The second strategy requires information about the structure of the natural ligand for a receptor. Development of new ligands then is based on the deliberate synthesis of specific analogs of the natural ligand in the hope of discovering a ligand that retains or has increased affinity for the receptor, together with bioavailability, stability, and other properties desired for a human pharmaceutical.
The third strategy requires information about the structure of the receptor itself. With this information, ligands are designed by the design of structures that are complementary to the binding site of the ligand.
The deficiencies of these three approaches are well known to those familiar with the art. Random input screening often requires examination of thousands of compounds before a single ligand has a chance of being identified. Analogs of the natural ligands often resemble the natural ligand in terms of bioavailability, stability, or other properties; often, these properties are undesirable in a human pharmaceutical. Further, while tremendous strides have been made in the science of molecular recognition over the past decade, it is still not possible to design a ligand for a receptor even given a high resolution experimental structure for the receptor itself.
One approach suggested for solving these problems comes under the title of xe2x80x9cin vitro selectionxe2x80x9d. The approach has several implementations. Most commonly, a collection, or library, of oligonucleotides of random sequence is presented to a receptor, often attached to a solid support. The receptor binds to only a few oligonucleotides in the library. The oligonucleotides in the pool that do not bind to the receptor are then washed from the receptor. The oligonucleotides that bind to the receptor tightly are then eluted from the receptor and recovered. These are then amplified by polymerase chain reaction technology, well known in the art (Mullis et al., U.S. Pat. No. 4,683,202), to yield a library of oligonucleotides whose members have a higher affinity, on average, than the members of the original pool. This new library is then subjected to mutation by methods well known in the art to create a new library of oligonucleotides with structures randomized around those of the starting library with increased affinity for the receptor. These are then subjected to the binding, elution, and amplification steps in repeated cycles, leading to oligonucleotides with increased binding activity. After several rounds of selection, a secondary library can next be prepared from a mixture of oligonucleotides already enriched with those that have affinity for a receptor, by amplifying the mixture using polymerases under conditions where the polymerase makes mistakes. The secondary library re-diversifies the library of ligands that already has some affinity to the receptor, permitting the in vitro selection experiment to search a region of combinatorial xe2x80x9csequence spacexe2x80x9d (Benner, S. A., Ellington, A. D. (1988) CRC Crit. Rev. Biochem. 23, 369-426) that is likely to contain oligonucleotides with the desired properties.
Those of ordinary skill in the art understand both the value of in vitro selection, and how to perform its individual steps in the laboratory. Preparation of mixtures of oligonucleotides, affinity purification of ligands from a mixture containing both ligands and non-ligands, amplification of oligonucleotides by PCR, and the mutagenesis of oligonucleotides are all well known in the art. In vitro selection has been recognized as being useful for obtaining ligands, receptors, and catalysts by many groups. Early articles by Joyce (Joyce, G. F. (1989) in RNA: Catalysis, Splicing, Evolution, Belfort and Shub, eds. Elsevier, Amsterdam, pp. 83-87), Irvine et al. (Irvine, D., Tuerk, C., Gold, L. (1991) J. Mol. Biol. 222, 739-761), and Szostak (Szostak, J. W. (1992) Trends Biochem. Soc. 17, 89-93) layed out the elements of classical in vitro selection methodologies. For example, in vitro selection was used to obtain oligonucleotides as ligands for reverse transcriptase by Chen et al. (Chen, H., Gold, L. (1994) Biochemistry 33, 8746-8756). In the patent literature, Gold and Tuerk (U.S. Pat. No. 5,270,163) propose in vitro selection methods on RNA libraries as an approach towards obtaining ligands and receptors. Gold (U.S. Pat. No. 5,476,766) propose an in vitro selection system for amplifying RNA to create ligands for thrombin. Jayasena and Gold (U.S. Pat. No. 5,472,841) consider oligonucleotide libraries where the 2xe2x80x2-hydroxyl group of the ribose ring of standard oligonucleotides may be replaced by a 2xe2x80x2-amino group. In principle, when selection methods permit enrichment of oligonucleotides that bind to a transition state analog, catalysts might also be obtained by in vitro selection (U.S. Pat. No. 5,270,163).
It is clear that in vitro selection based on natural oligonucleotides has limited efficacy, however. As discussed in Ser. No. 07/594,290, filed: Oct. 9, 1990, now issued as U.S. Pat. No. 5,432,272, of which the instant application is a continuation-in-part, the primary difficulty with in vitro selection as a tool for creating molecules with desired properties arises from the fact that natural oligonucleotides do not themselves display a wide diversity in either structure, or conformation, or functionality. Oligonucleotides that form the libraries used in classical in vitro selection experiments are built from only four building blocks. These building blocks carry relatively little useful functionality, especially compared to that carried by proteins. Thus, oligonucleotides with tight affinity for some receptors occur only infrequently in a population of random oligonucleotides. This makes it difficult to identify oligonucleotides within a library of standard oligonucleotide sequences that have satisfactory binding or catalytic properties for a range of ligands and reactions.
The instant invention provides improvements to classical in vitro selection methods already known in the art, to increase the effectiveness of in vitro selection experiments as a tool for generating new structures (e.g., bent oligonucleotides), ligands, receptors, and catalysts. The improvements comprise incorporating into the oligonucleotide analogs that are the components of a library to which in vitro selection methods functionalized standard nucleotides and non-standard nucleotides, both functionalized and not functionalized, and incorporating functionalized cofactors into the in vitro selection experiment.